Anticancer compounds and methods

ABSTRACT

The testing of tumor cells, including human tumors capable of metastases, in assays employing fibronectin-depleted substrates is described. Ex vivo induction of cells, including biopsied human cells, is performed with invasion-inducing agents. Additionally, anti-cancer chemotherapeutics are described. Specifically, chemotherapeutic agents which have anti-metastatic and anti-growth properties are described including non-peptide compositions of matter.

This is a Continuation Application of Ser. No. 08/915,189 filed on Aug.20, 1997 which is a Continuation-In-Part of Ser. No. 08/754,322 whichsubsequently issued as U.S. Pat. No. 5,840,514 on Nov. 24, 1998.

FIELD OF THE INVENTION

The present invention relates to the treatment of cancer, to the testingof cancer cells for their ability to invade tissues and causemetastases, and to the identification and use of drugs to inhibit tumorinvasion and growth.

BACKGROUND

The term “chemotherapy” simply means the treatment of disease withchemical substances. The father of chemotherapy, Paul Ehrlich, imaginedthe perfect chemotherapeutic as a “magic bullet”; such a compound wouldkill invading organisms without harming the host. This targetspecificity is sought in all types of chemotherapeutics, includinganticancer agents.

However, specificity has been the major problem with anticancer agents.In the case of anticancer agents, the drug needs to distinguish betweenhost cells that are cancerous and host cells that are not cancerous. Thevast bulk of anticancer drugs are indiscriminate at this level.Typically anticancer agents have negative hematological effects (e.g.,cessation of mitosis and disintegration of formed elements in marrow andlymphoid tissues), and immunosuppressive action (e.g., depressed cellcounts), as well as a severe impact on epithelial tissues (e.g.,intestinal mucosa), reproductive tissues (e.g., impairment ofspermatogenesis), and the nervous system. P. Calabresi and B. A.Chabner, In: Goodman and Gilman The Pharmacological Basis ofTherapeutics (Pergamon Press, 8th Edition) (pp. 1209-1216).

Success with chemotherapeutics as anticancer agents has also beenhampered by the phenomenon of multiple drug resistance, resistance to awide range of structurally unrelated cytotoxic anticancer compounds. J.H. Gerlach et al., Cancer Surveys, 5:25-46 (1986). The underlying causeof progressive drug resistance may be due to a small population ofdrug-resistant cells within the tumor (e.g., mutant cells) at the timeof diagnosis. J. H. Goldie and Andrew J. Coldman, Cancer Research,44:3643-3653 (1984). Treating such a tumor with a single drug firstresults in a remission, where the tumor shrinks in size as a result ofthe killing of the predominant drug-sensitive cells. With thedrug-sensitive cells gone, the remaining drug-resistant cells continueto multiply and eventually dominate the cell population of the tumor.

Finally, the treatment of cancer has been hampered by the fact thatthere is considerable heterogeneity even within one type of cancer. Somecancers, for example, have the ability to invade tissues and display anaggressive course of growth characterized by metastases. These tumorsgenerally are associated with a poor outcome for the patient. And yet,without a means of identifying such tumors and distinguishing suchtumors from non-invasive cancer, the physician is at a loss to changeand/or optimize therapy.

What is needed is a specific anticancer approach that is reliable for awide variety of tumor types, and particularly suitable for invasivetumors. Importantly, the treatment must be effective with minimal hosttoxicity.

SUMMARY OF THE INVENTION

The present invention relates to the treatment of cancer, to the testingof cancer cells for their ability to invade tissues and causemetastases, and to the identification and use of drugs to inhibit tumorinvasion and growth. The present invention provides: A) an in vitromodel for testing cancer cells and evaluating invasive potential; B) ascreening assay for identifying drugs that inhibit tumor invasion; andC) chemotherapeutics for treating cancer.

A variety of assay formats are contemplated for testing the invasivepotential of cancer cells. In one embodiment, a portion of a patient'stumor is obtained (e.g., by biopsy) and placed in tissue culture on afibronectin-free substrate. Thereafter, the response of the tumor cellsto fibronectin or a fibronectin-derived peptide is assessed. Wherefibronectin induces invasion of the membrane, the tumor can beconsidered to have metastatic potential. Where there is no significantinvasion of the membrane, the tumor can be considered (at that time) tobe non-metastatic.

In one embodiment, the present invention contemplates a method ofevaluating human cancer comprising: a) providing: i) a human cancerpatient, ii) a fibronectin-free substrate, and iii) one or moreinvasion-inducing agents; b) obtaining cancer cells from said patient;c) contacting said cells ex vivo with said fibronectin-free substrateand one or more invasion-inducing agents; and d) detecting cancer cellinvasion of said substrate. Preferably the cancer cells are cultured inserum-free culture media so as to essentially avoid introducingcomplicating factors. In one embodiment, the invasion-inducing agent isa peptide, said peptide comprising the sequence PHSRN (SEQ ID NO:1). Ina preferred embodiment the invasion inducing agent is intactfibronectin.

While not limited to any mechanism, it is believed that cells exposed toinvasion-inducing agents in this manner are potentially rendered capableof invading the substrate. Indeed, the present invention contemplatesstimulation of invasion by all cells of the body, including, but notlimited to: epithelial (keratinocytes, mammary and prostate epithelial),connective tissue (fibroblasts), and muscle (myoblast) cells. Again,while not limited to any mechanism, it is believed that the invasioninducing agent comprising the sequence PHSRN (SEQ ID NO:1) binds to theα5μ1 receptor on the cancer cell and thereby induces invasion of thesubstrate. In this regard, the present invention provides a method oftesting human cancer cells comprising: a) providing: i) a human cancerpatient, ii) a fibronectin-free substrate, and iii) one or moreinvasion-inducing agents; b) obtaining α5μ1 integrin fibronectinreceptor-expressing cancer cells from said patient; c) culturing saidcells in serum-free culture media on said substrate in the presence ofsaid invasion-inducing agents; and d) detecting cancer cell invasion ofsaid substrate.

As noted above, the present invention also contemplates a screeningassay for identifying drugs that inhibit tumor invasion. The presentinvention contemplates a screening assay utilizing the binding activityof fibronectin-derived peptides. In one embodiment, an inducible tumorcell line is placed in tissue culture on a fibronectin-free substrate.Thereafter, as an inducible tumor cell line, the tumor will be induced(under ordinary conditions) by fibronectin or the fibronectin-derivedpeptide to invade the substrate. However, in this drug screening assay,candidate drug inhibitors are added to the tissue culture (this can bedone individually or in mixtures). Where the inducible tumor cell isfound to be inhibited from invading the substrate, a drug inhibitor isindicated.

It is not intended that the present invention be limited by the natureof the drugs screened in the screening assay of the present invention. Avariety of drugs, including peptides, are contemplated.

Finally, the present invention contemplates chemotherapeutics fortreating invasive tumors. Specifically, a variety of anti-invasivechemotherapeutic agents are contemplated to antagonize theinvasion-promoting activity of the PHSRN (SEQ ID NO:1) peptide. In thepreferred embodiment, the anti-invasive agent is a peptide with theamino acid sequence PHSCN (SEQ ID NO:86). In another embodiment, theanti-invasive agent is a peptide which has an amino acid sequencecomprising a sequence selected from the group consisting of CHSRN (SEQID NO:87), PCSRN (SEQ ID NO:88), PHCRN (SEQ ID NO:89), and PHSRC (SEQ IDNO:90). In another embodiment, the anti-invasive agent is a peptidewhich has an amino acid sequence comprising PHSXN (SEQ ID NO:91), whereX is an amino acid selected from the group consisting of homo-cysteine,the D-isomer of cysteine, histidine, or penicillamine.

The present invention also contemplates an anti-invasive agentcomprising the amino acid sequence X₁HSX₂N (SEQ ID NO:92), wherein X₁ iseither proline, histidine, or not an amino acid, and X₂ is an amino acidselected from the group consisting of the L-isomer of cysteine, theD-isomer of cysteine, homo-cysteine, histidine, or penicillamine. Inanother embodiment, the present invention contemplates an anti-invasiveagent comprising the amino acid sequence X₁X₂X₃X₄X₅ (SEQ ID NO:93),wherein X₁ is an amino acid selected from the group consisting ofproline, glycine, valine, histidine, isoleucine, phenylalanine,tyrosine, and tryptophan, and X₂ is an amino acid selected from thegroup consisting of histidine, proline, tyrosine, asparagine, glutamine,arginine, lysine, phenylalanine, and tryptophan, and X₃ is an amino acidselected from the group consisting of serine, threonine, alanine,tyrosine, leucine, histidine, asparagine, and glutamine, and X₄ is anamino acid selected from the group consisting of cysteine,homo-cysteine, penicillamine, histidine, tyrosine, asparagine,glutamine, and methionine, and X₅ is an amino acid selected from thegroup consisting of asparagine, glutamine, serine, threonine, histidine,and tyrosine. In the preferred embodiment the peptide is PHSCN (SEQ IDNO:86), where the cysteine is the L-isomer.

It is further contemplated that the anti-invasive agents named abovecomprise the named amino acid sequence and additional amino acids addedto the amino terminus, the carboxyl terminus, or both the amino andcarboxyl termini. In one embodiment, the anti-invasive agent is up tofive hundred amino acids in length. It is also contemplated that, insome embodiments, the anti-invasive agents named above comprise apeptide with the amino terminus blocked by standard methods to preventdigestion by exopeptidases, for example by acetylation; and the carboxylterminus blocked by standard methods to prevent digestion byexopeptidases, for example, by amidation.

In this regard, the present invention provides a method of treatingcancer comprising: a) providing: i) a subject having cancer, and ii) acomposition of matter comprising a peptide which inhibits the tumorinvasion-promoting activity of the PHSRN (SEQ ID NO:1) sequence ofplasma fibronectin; and b) administering said composition to saidsubject. The present invention further contemplates using antagonistsbefore and/or after surgical removal of the primary tumor. In oneembodiment, the method comprises administering a PHSRN (SEQ ID NO:1)antagonist as adjunct therapy with additional chemotherapeutics.

While not limited to any mechanism, it is believed that theseanti-invasive chemotherapeutic agents antagonize the invasion-promotingactivity of the PHSRN (SEQ ID NO:1) sequence (e.g., of fibronectin) byblocking the binding of this sequence to its receptor on tumor cells.Again, while not limited to any mechanism, it is believed that the PHSRN(SEQ ID NO:1) sequence may promote invasion by acting to displace adivalent cation (Mg+2, Ca+2, or Mn+) in the α5μ1 receptor on metastatictumor cells, and the above named chemotherapeutic anti-invasive agentsmight act to inhibit this invasion by chelating one or more of thesedivalent cations.

In another embodiment, the present invention contemplates anti-invasionantagonists to the IKVAV (SEQ ID NO:2) sequence of laminin, includingbut not limited to, peptides comprising the structure, ICVAV (SEQ IDNO:94), and corresponding peptide mimetics.

DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows the one embodiment of the substrate usedaccording to the present invention for testing tumor cells. The spatialrelationship of the ectoderm of the Strongylocentrotus purpuratus embryoto its extracellular matrix and to blastocoelar structures are shown (s,spicules; h, hyalin layer; e, ectoderm; b, subectodermal basementmembrane; bl, blastocoel; g, stomach of the primitive gut; c, coelomicpouches). The esophagus and intestine do not appear on the side of theembryo shown.

FIG. 2 is a graph showing the results of the testing of tumor cells onfibronectin-containing substrates and fibronectin-depleted substrates invitro without the use of the invasion-inducing agents of the presentinvention.

FIG. 3 is a graph showing the results of the testing of tumor cells onfibronectin-depleted substrates in vitro with and withoutinvasion-inducing agents according one embodiment of the method of thepresent invention.

FIG. 4 is a graph showing the results of the testing of normal cells onfibronectin-depleted substrates in vitro with and withoutinvasion-inducing agents according one embodiment of the method of thepresent invention.

FIG. 5A is a graph showing the results of inhibiting serum-induced humanbreast cancer cell invasion of the SU-ECM substrate with varyingconcentrations of the PHSCN (SEQ ID NO:86) peptide.

FIG. 5B is a graph showing the results of inhibiting PHSRN (SEQ IDNO:88)-induced invasion by both human breast cancer cells and normalhuman mammary epithelial cells of the SU-ECM substrate with varyingconcentrations of the PHSCN (SEQ ID NO:86) peptide.

FIG. 6A is a graph showing the results of inhibiting serum-induced humanprostate cancer cell invasion of the SU-ECM substrate with varyingconcentrations of the PHSCN (SEQ ID NO:86) peptide.

FIG. 6B is a graph showing the results of inhibiting PHSRN-inducedinvasion by both human prostate cancer cells and normal prostateepithelial cells of the SU-ECM substrate with varying concentrations ofthe PHSCN (SEQ ID NO:86) peptide.

FIG. 7A is a graph showing the results of testing serum-induced ratprostate cancer cell invasion of the SU-ECM substrate with and withoutthe PHSCN (SEQ ID NO:86) peptide.

FIG. 7B is a graph showing the results of inhibiting PHSRN-induced (SEQID NO:1) rat prostate cancer cell invasion of the SU-ECM substrate withvarying concentrations of the PHSCN (SEQ ID NO:86) peptide.

FIG. 8 is a graph showing the results of inhibiting serum-induced ratprostate cancer cell invasion of the SU-ECM substrate with varyingconcentrations of the PHS(homo)CN (SEQ ID NO:85) peptide.

FIG. 9A is a graph showing the results of testing tumor growth in ratsinjected with prostate cancer cells, with half of the rats receivingtreatment with the PHSCN (SEQ ID NO:86) peptide, initiated inconjunction with the initial injection.

FIG. 9B is a graph showing the results of determining the mean number oflung metastases in the two groups of rats described in FIG. 9 a.

FIG. 10A is a graph showing the results of testing tumor growth in ratsinjected with prostate cancer cells, with half of the rats receivingtreatment with the PHSCN (SEQ ID NO:86) peptide, initiated 24 hoursafter the initial cancer cell injection.

FIG. 10B is a graph showing the results of determining the mean numberof lung metastases in the two groups of rats described in FIG. 10 a.

FIG. 10C is a graph showing the results of determining the mean mass ofintraperitoneal metastatic tissues in the two groups of rats describedin FIG. 10 a.

DEFINITIONS

The term “drug” as used herein, refers to any medicinal substance usedin humans or other animals. Encompassed within this definition arecompound analogs, naturally occurring, synthetic and recombinantpharmaceuticals, hormones, antimicrobials, neurotransmitters, etc.

The term “inducing agent” refers to any compound or molecule which iscapable of causing (directly or indirectly) the invasion of cells in asubstrate. “Inducing agents” include, but are not limited to,PHSRN-containing (SEQ ID NO:1) peptides and related peptides (seebelow).

The term “receptors” refers to structures expressed by cells and whichrecognize binding molecules (e.g., ligands).

The term “antagonist” refers to molecules or compounds which inhibit theaction of a “native” or “natural” compound (such as fibronectin).Antagonists may or may not be homologous to these natural compounds inrespect to conformation, charge or other characteristics. Thus,antagonists may be recognized by the same or different receptors thatare recognized by the natural compound. “Antagonists” include, but arenot limited to, PHSCN-containing (SEQ ID NO:86) peptides and relatedpeptides (see below).

The term “host cell” or “cell” refers to any cell which is used in anyof the screening assays of the present invention. “Host cell” or “cell”also refers to any cell which either naturally expresses particularreceptors of interest or is genetically altered so as to produce thesenormal or mutated receptors.

The term “chemotherapeutic agent” refers to molecules or compounds whichinhibit the growth or metastasis of tumors. “Chemotherapeutics” include,but are not limited to, PHSCN-containing (SEQ ID NO:86) peptides andrelated peptides (see below).

As noted above, the present invention contemplates both the D and Lisomers of cysteine which are identified collectively as “C”.

The present invention also contemplates homo-cysteine, which isidentified as “hC”.

DESCRIPTION OF THE INVENTION

The present invention generally relates to the treatment of cancer, andmore specifically, to the testing of cancer cells for their ability toinvade tissues and cause metastases, and to the identification and useof drugs to inhibit tumor invasion and growth. As a prelude tometastasis, it is believed that cancer cells proteolytically alterbasement membranes underlying epithelia or the endothelial linings ofblood and lymphatic vessels, invade through the defects created byproteolysis, and enter the circulatory or lymphatic systems to colonizedistant sites. During this process, the secretion of proteolytic enzymesis coupled with increased cellular motility and altered adhesion. Aftertheir colonization of distant sites, metastasizing tumor cellsproliferate to establish metastatic nodules.

As noted above, chemotherapeutic agents are currently employed to reducethe unrestricted growth of cancer cells, either prior to surgicalremoval of the tumor (neoadjuvant therapy) or after surgery (adjuvanttherapy). However, none of these methods has proved curative oncemetastasis has occurred. Since unrestricted invasive behavior is also ahallmark of metastatic tumor cells, methods for directly inhibitingtumor cell invasion and metastasis are needed.

A. Assays for Testing Tumor Invasion

Discovering how to inhibit the invasive behavior of tumor cells tointervene in the metastatic cascade first requires the development ofassays with which to test tumor cell invasion in vitro. Two assaysystems are contemplated for use in the method of the present inventionto test the tumor cell invasion.

1. Fibronectin-Depleted Substrates

In one assay system, the present invention contemplates usingfibronectin-depleted substrates. These are substrates that originallycontain fibronectin that are treated according to the methods of thepresent invention (see below) to remove fibronectin. It is not intendedthat the present invention be limited by the nature of the originalsubstrate; such fibronectin-containing substrates suitable for treatmentand depletion include: i) complex substrates containing a variety ofextracellular proteins and ii) less complex substrates containingfibronectin along with one or two other proteins (e.g. collagen,laminin, etc.).

It is also not intended that the present invention be limited by theprecise amount of fibronectin remaining after the substrate has beentreated. In other words, while the methods of the present inventionremove fibronectin, and in some embodiments, remove substantially allfibronectin, it is within the meaning of the term “fibronectin-depleted”substrate that a small amount of fibronectin remain in the substrate.

In one embodiment, the present invention contemplates using anextracellular matrix available commercially. For example, the presentinvention contemplates treating basement membrane matrices such as ECMGEL, a matrix from mouse sarcoma (commercially available from Sigma, St.Louis, Mo.). However, it is not intended that the present invention belimited by the particular fibronectin-containing substrate. For example,other commercially available substrates are contemplated, such as thecommonly used substrate Matrigel (available from Becton DickinsonLabware, Catalog #40234); Matrigel can be treated appropriatelyaccording to the methods of the present invention so as to render it“fibronectin-depleted” (see below). Untreated Matrigel (and similarsubstrates) have been used to demonstrate the importance of proteasesand motility factors in the invasion and metastasis of many tumors.However, these invasion substrates are not available as serum-freesubstrates; thus, the regulation of tumor cell invasive behavior byserum components, such as plasma fibronectin, is a complicating factorwith untreated Matrigel.

Consequently, the present invention contemplates a fibronectin-freesubstrate. In this embodiment, Matrigel is treated so that it issubstantially fibronectin-free. The preparation of fibronectin-freeMatrigel involves “panning” the Matrigel substrate on gelatin as well as“panning” the substrate on anti-fibronectin antibody (anti-humanfibronectin IgG is available commercially, such as antibody from PromegaCorporation, Madison, Wis.).

2. Naturally Occurring Fibronectin-Free Substrates

In another embodiment, the present invention contemplates substratesthat are naturally free of fibronectin; such a source provides, forexample, basement membranes permeable to select types of normallyinvasive cells, such membranes being naturally serum-free. In oneembodiment, the present invention contemplates sea urchins as a sourceof such membranes. In this regard, the ectoderm of sea urchin embryos isone cell thick, and secretes an underlying basement membrane (seeFIG. 1) very similar to that of mammals. These embryos contain nocirculatory or lymphatic systems; and thus, their basement membranes areserum-free. In embryos, the subectodermal basement membrane functionssimultaneously as a migration substrate for several, specificmesenchymal cell types while it functions as an invasion substrate forothers. Sea urchin embryo basement membranes (SU-ECM) can be prepared bymild detergent treatment as described in D. Livant et al., CancerResearch 55:5085 (1995) and described in the Experimental section below.

Regardless of which of the two types of substrates are employed, theinvasion substrates of the present invention are easy to prepare andgive rapid, highly consistent results with a variety of cells,including: a) cell lines from: i) primary and metastatic tumors, and ii)normal epithelial tissues; as well as b) cells from primary tissuesamples of both tumors, their surrounding normal tissues, and neonatalmelanocytes, fibroblasts, and keratinocytes from circumcised tissue.

In one embodiment, the present invention contemplates a method ofevaluating human cancer comprising: a) providing: i) a human cancerpatient (such as a patient with breast cancer or prostate cancer), ii) afibronectin-free substrate (for example, a fibronectin-depletedsubstrate) and iii) one or more invasion-inducing agents (discussedbelow); b) obtaining cancer cells from said patient (such as from abiopsy); c) contacting said cells ex vivo (i.e., outside the body) withsaid fibronectin-free substrate and said one or more invasion-inducingagents; and d) measuring the extent of cancer cell invasion of saidsubstrate. Preferably the cancer cells are cultured in serum-freeculture media so as to avoid introducing complicating factors.

3. Inducing Agents

It is not intended that the present invention be limited by the natureof the agent that causes or induces cells to invade the fibronectin-freesubstrates of the present invention. Such agents can be identifiedfunctionally by simply adding them to the cell culture and measuring theextent of invasion.

In one embodiment, the invasion-inducing agent comprises a peptidederived from fibronectin. In a preferred embodiment, the invasioninducing agent is intact fibronectin.

While not limited to any mechanism, it is believed that cells exposed toinvasion-inducing agents in this manner are potentially rendered capableof invading the substrate. Again, while not limited to any mechanism, itis believed that the invasion inducing agent comprising the sequencePHSRN (SEQ ID NO:1) binds to the α5μ1 receptor on the cancer cell andthereby induces invasion of the substrate. In this regard, the presentinvention provides a method of treating cells comprising: a) providing:i) cells expressing the α5β1 receptor, ii) a fibronectin-free substrate,and iii) one or more invasion-inducing agents; b) culturing said cellsin serum-free culture media on said substrate in the presence of saidinvasion-inducing agents; and d) measuring the extent of cell invasionof said substrate. In one embodiment, the cells are normal epithelialcells or fibroblasts. In another embodiment, the cells are human cancercells.

B. Drug Screening Assays

The present invention also contemplates a screening assay foridentifying drugs that inhibit tumor invasion. The present inventioncontemplates a screening assay (in the presence and absence of serum)utilizing the binding activity of fibronectin-derived peptides. In oneembodiment, an inducible tumor cell line is placed in tissue culture ona fibronectin-free substrate. The tumor cells will be induced (underordinary conditions) by the fibronectin-derived peptide to invade thesubstrate.

In one embodiment, the invasion-inducing agent comprises a peptidederived from fibronectin. In a preferred embodiment, said peptidecomprises the sequence PHSRN (SEQ ID NO:1). Of course, the peptide maybe larger than five amino acids; indeed, the peptide fragment offibronectin may contain hundreds of additional residues (e.g., fivehundred amino acids). One such larger peptide is set forth in U.S. Pat.No. 5,492,890 (hereby incorporated by reference). In one embodiment, thePHSRN-containing (SEQ ID NO:1) peptide is less than one hundred aminoacids in length and lacks the RGD (SEQ ID NO:81) sequence characteristicof fibronectin. A variety of PHSRN-containing (SEQ ID NO:1) peptides arecontemplated, including the PHSRN (SEQ ID NO:1) peptide itself andrelated peptides where additional amino acids are added to the carboxylterminus, including (but not limited to) peptides comprising thesequence: 1) PHSRN (SEQ ID NO:1), 2) PHSRNS (SEQ ID NO:3), 3) PHSRNSI(SEQ ID NO:4), 4) PHSRNSIT (SEQ ID NO:5), 5) PHSRNSITL (SEQ ID NO:6), 6)PHSRNSITLT (SEQ ID NO:7), 7) PHSRNSITLTN (SEQ ID NO:8), 8) PHSRNSITLTNL(SEQ ID NO:9), 9) PHSRNSITLTNLT (SEQ ID NO:10), 10) PHSRNSITLTNLTP (SEQID NO:11), and 11) PHSRNSITLTNLTPG (SEQ ID NO:12). Alternatively,PHSRN-containing (SEQ ID NO:1) peptides are contemplated where aminoacids are added to the amino terminus, including (but not limited to)peptides comprising the sequence: 1) PEHFSGRPREDRVPHSRN (SEQ ID NO:13),2) EHFSGRPREDRVPHSRN (SEQ ID NO:14), 3) HFSGRPREDRVPHSRN (SEQ ID NO:15),4) FSGRPREDRVPHSRN (SEQ ID NO:16), 5) SGRPREDRVPHSRN (SEQ ID NO:17), 6)GRPREDRVPHSRN (SEQ ID NO:18), 7) RPREDRVPHSRN (SEQ ID NO:19), 8)PREDRVPHSRN (SEQ ID NO:20), 9) REDRVPHSRN (SEQ ID NO:21), 10) EDRVPHSRN(SEQ ID NO:22), 11) DRVPHSRN (SEQ ID NO:23), 12) RVPHSRN (SEQ ID NO:24),and 13) VPHSRN (SEQ ID NO:25). Finally, the present inventioncontemplates PHSRN-containing (SEQ ID NO:1) peptides where amino acidsare added to both the amino and carboxyl termini, including (but notlimited to) peptides comprising the sequencePEHFSGRPREDRVPHSRNSITLTNLTPG (SEQ ID NO:26), as well as peptidescomprising portions or fragments of the PHSRN-containing (SEQ ID NO:1)sequence PEHFSGRPREDRVPHSRNSITLTNLTPG (SEQ ID NO:26).

Peptides containing variations on the PHSRN (SEQ ID NO:1) motif arecontemplated. For example, the present invention also contemplatesPPSRN-containing (SEQ ID NO:27) peptides for use in the above-namedassays. Such peptides may vary in length in the manner described abovefor PHSRN-containing (SEQ ID NO:1) peptides. Alternatively, PPSRN (SEQID NO:27) may be used as a peptide of five amino acids.

Similarly, peptides comprising the sequence -HHSRN- (SEQ ID NO:28),-HPSRN- SEQ ID NO:29), -PHTRN- (SEQ ID NO:30) -HHTRN- (SEQ ID NO:31),-HPTRN- (SEQ ID NO:32), -PHSNN- (SEQ ID NO:33), -HHSNN- (SEQ ID NO:34),-HPSNN- (SEQ ID NO:35), -PHTNN- (SEQ ID NO:36), -HHTNN- (SEQ ID NO:37),-HPTNN- (SEQ ID NO:38), -PHSKN- (SEQ ID NO:39), -HHSKN- (SEQ ID NO:40),-HPSKN- (SEQ ID NO:41), -PHTKN- (SEQ ID NO:42), -HHTKN- (SEQ ID NO:43),-HPTKN- SEQ ID NO:44), -PHSRR- (SEQ ID NO:45), -HHSRR- (SEQ ID NO:46),-HPSRR- (SEQ ID NO:47), -PHTRR- (SEQ ID NO:48), -HHTRR- (SEQ ID NO:49),-HPTRR- (SEQ ID NO:50), -PHSNR- (SEQ ID NO:51), -HHSNR- (SEQ ID NO:52),-HPSNR- (SEQ ID NO:53), -PHTNR- (SEQ ID NO:54), -HHTNR- (SEQ ID NO:55),-HPTNR- (SEQ ID NO:56), -PHSKR- (SEQ ID NO:57), -HHSKR- (SEQ ID NO:58),-HPSKR- (SEQ ID NO:59), -PHTKR- (SEQ ID NO:60), -HHTKR- (SEQ ID NO:61),-HPTKR- (SEQ ID NO:62), -PHSRK- (SEQ ID NO:63), -HHSRK- (SEQ ID NO:64),-HPSRK- (SEQ ID NO:65), -PHTRK- (SEQ ID NO:66), -HHTRK- (SEQ ID NO:67),-HPTRK- (SEQ ID NO:68), -PHSNK- (SEQ ID NO:69), -HHSNK- (SEQ ID NO:70),-HPSNK- (SEQ ID NO:71), -PHTNK- (SEQ ID NO:72), -HHTNK- (SEQ ID NO:73),-HPTNK- (SEQ ID NO:74), -PHSKK- (SEQ ID NO:75), -HHSKK- (SEQ ID NO:76),-HPSKK- (SEQ ID NO:77), -PHTKK- (SEQ ID NO:78), -HHTKK- (SEQ ID NO:79),or -HPTKK- (SEQ ID NO:80) are contemplated by the present invention.Such peptides can be used as five amino acid peptides or can be part ofa longer peptide (in the manner set forth above for PHSRN-containing(SEQ ID NO:1) peptides).

In another embodiment, the present invention contemplates an inducingagent comprising the amino acid sequence X₁X₂X₃X₄X₅ (SEQ ID NO:93),wherein X₁ is an amino acid selected from the group consisting ofproline, glycine, valine, histidine, isoleucine, phenylalanine,tyrosine, and tryptophan, and X₂ is an amino acid selected from thegroup consisting of histidine, proline, tyrosine, asparagine, glutamine,arginine, lysine, phenylalanine, and tryptophan, and X₃ is an amino acidselected from the group consisting of serine, threonine, alanine,tyrosine, leucine, histidine, asparagine, and glutamine, and X₄ is anamino acid selected from the group consisting of arginine, lysine, andhistidine, and X₅ is an amino acid selected from the group consisting ofasparagine, glutamine, serine, threonine, histidine, and tyrosine.

In this drug screening assay, candidate drug inhibitors are added to thetissue culture (this can be done individually or in mixtures). Where theinducible tumor cell is found to be inhibited from invading thesubstrate, a drug inhibitor is indicated (see Examples section belowusing the PHSCN (SEQ ID NO:86) peptide).

It is not intended that the present invention be limited by the natureof the drugs screened in the screening assay of the present invention. Avariety of drugs, including peptides and non-peptide mimetics, arecontemplated.

It is also not intended that the present invention be limited by theparticular tumor cells used for drug testing. A variety of tumor cells(for both positive and negative controls) are contemplated (includingbut not limited to the cells set forth in Table 1 below).

C. Invasion-Inducing Agents and Antagonists

While an understanding of the mechanisms involved in metastatic canceris not necessary to the successful practice of the present invention, itis believed that tumor cell invasion of basement membranes occurs atseveral points in the metastatic cascade: (1) when epithelial tumorcells (such as those of breast and prostate cancers) leave theepithelium and enter the stroma, (2) when tumor cells enter thecirculatory or lymphatic systems, and (3) when tumor cells leave thecirculatory or lymphatic systems to invade distant sites. Thus,intervention in the induction of tumor cell invasiveness by using aPHSRN (SEQ ID NO:1) antagonist, such as the PHSCN (SEQ ID NO:86)peptide, to block tumor cell receptors for this sequence is contemplatedas a method for decreasing the rate of metastasis.

One advantage of this strategy is that leukocytes are the only normalcells known to invade tissues to carry out their functions, andrelatively few leukocytes are invasive at a given time. Thus, relativelysmall doses of an anti-invasion antagonist which blocks the binding ofPHSRN (SEQ ID NO:1) to its receptor are required. Also, other than someimmunodepression, there should be relatively few side effects associatedwith anti-metastatic treatment using compounds designed to block theinduction of invasion. The lack of debilitating side effects expectedfrom anti-invasive therapy means that using it in combination withanti-proliferative agents would be uncomplicated, and that it could beused prior to surgery or even prophylactically to block tumor cellinvasion and metastasis.

The IKVAV (SEQ ID NO:2) sequence of laminin, a prevalent insolubleprotein of the extracellular matrix, is known to stimulate livercolonization by metastatic human colon cancer cells in athymic mice [seeBresalier et al., Cancer Research 55:2476 (1995)]. Since IKVAV (SEQ IDNO:2), like PHSRN (SEQ ID NO:1), contains a basic amino acid (K) which,by virtue of its positive charge, might also function to displace adivalent cation from its integrin receptor and stimulate invasion, thepresent invention contemplates applying the strategy of developinganti-invasion antagonists to the IKVAV (SEQ ID NO:2) sequence oflaminin.

TABLE 1 Designation And Origin Of Human Cell Lines And Strains¹ OriginCell Lines or Strains Colonic carcinoma SW1116, HCT116, SKCO-1, HT-29,KM12C, KM12SM, KM12L4, SW480 Pancreatic carcinoma BxPC-3, AsPC-1,Capan-2, MIA PaCa-2, Hs766T Colon adenoma VaCo 235 Lung carcinoma A549Prostate carcinoma PC-3, DU-145 Breast carcinoma 009P, 013T, SUM-52 PELymphoma Daudi, Raji Breast epithelium 006FA Diploid fibroblast HCS(human corneal stroma), MRC-5 ¹The SW1116, HT-29, SW480, Rajilymphoblastoid cells, and the pancreatic lines are obtained from theAmerican Type Culture Collection.

1. Antagonists

It is not intended that the present invention be limited by the natureof the agent that inhibits tumor invasiveness. A variety ofanti-invasive chemotherapeutics are contemplated to antagonize theinvasion-promoting activity of the PHSRN (SEQ ID NO:1) sequence.

In the preferred embodiment, the anti-invasive agent is a peptide withthe amino acid sequence PHSCN (SEQ ID NO:86). In another embodiment, theanti-invasive agent is a peptide which has an amino acid sequencecomprising a sequence selected from the group consisting of CHSRN (SEQID NO:87), PCSRN (SEQ ID NO:88), PHCRN (SEQ ID NO:89), and PHSRC (SEQ IDNO:90). In another embodiment, the anti-invasive agent is a peptidewhich has an amino acid sequence comprising PHSXN (SEQ ID NO:91), whereX is an amino acid selected from the group consisting of homo-cysteine,the D-isomer of cysteine, histidine, or penicillamine.

The present invention also contemplates an anti-invasive agentcomprising the amino acid sequence X₁HSX₂N (SEQ ID NO:92), wherein X₁ iseither proline, histidine, or not an amino acid, and X₂ is an amino acidselected from the group consisting of the L-isomer of cysteine, theD-isomer of cysteine, homo-cysteine, histidine, or penicillamine. Inanother embodiment, the present invention contemplates an anti-invasiveagent comprising the amino acid sequence X₁X₂X₃X₄X₅ (SEQ ID NO:93),wherein X₁ is an amino acid selected from the group consisting ofproline, glycine, valine, histidine, isoleucine, phenylalanine,tyrosine, and tryptophan, and X₂ is an amino acid selected from thegroup consisting of histidine, proline, tyrosine, asparagine, glutamine,arginine, lysine, phenylalanine, and tryptophan, and X₃ is an amino acidselected from the group consisting of serine, threonine, alanine,tyrosine, leucine, histidine, asparagine, and glutamine, and X₄ is anamino acid selected from the group consisting of cysteine,homo-cysteine, penicillamine, histidine, tyrosine, asparagine,glutamine, and methionine, and X₅ is an amino acid selected from thegroup consisting of asparagine, glutamine, serine, threonine, histidine,and tyrosine. In the preferred embodiment the peptide is PHSCN (SEQ IDNO:86), where the cysteine is the L-isomer.

Similarly, peptides comprising the sequence -PSCN- (SEQ ID NO:102),-HSCN- (SEQ ID NO:96), -PSCN- (SEQ ID NO: 102), -HTCN- (SEQ ID NO:99),-PTCN- (SEQ ID NO:105), -HSCN- (SEQ ID NO:96), -HSCN- (SEQ ID NO:96),-PSCN- (SEQ ID NO:102), -HTCN- (SEQ ID NO:99), -HTCN- (SEQ ID NO:99),-PTCN- (SEQ ID NO:105), -HSCN- (SEQ ID NO:96), -HSCN- (SEQ ID NO:96),-PSCN- (SEQ ID NO: 102), -HTCN- (SEQ ID NO:99), -HTCN- (SEQ ID NO:99),-PTCN- (SEQ ID NO:105), -HSCR- (SEQ ID NO:97), -HSCR- (SEQ ID NO:97),-PSCR- (SEQ ID NO:103), -HTCR- (SEQ ID NO:100), -HTCR- (SEQ ID NO:100),-PTCR- (SEQ ID NO:106), -HSCR- (SEQ ID NO:97), -HSCR- (SEQ ID NO:97),-PSCR- (SEQ ID NO:103), -HTCR- (SEQ ID NO:100), -HTCR- (SEQ ID NO:100),-PTCR- (SEQ ID NO:106), -HSCR- (SEQ ID NO:97), -HSCR- (SEQ ID NO:97),-PSCR- (SEQ ID NO:103), -HTCR- (SEQ ID NO:100), -HTCR- (SEQ ID NO:100),-PTCR- (SEQ ID NO:106), -HSCK- (SEQ ID NO:95), -HSCK- (SEQ ID NO:95),-PSCK- (SEQ ID NO:101), -HTCK- (SEQ ID NO:98), -HTCK- (SEQ ID NO:98),-PTCK- (SEQ ID NO:104), -HSCK- (SEQ ID NO:95), -HSCK- (SEQ ID NO:95),-PSCK- (SEQ ID NO:101), -HTCK- (SEQ ID NO:98), -HTCK- (SEQ ID NO:98),-PTCK- (SEQ ID NO:104), -HSCK- (SEQ ID NO:95), -HSCK- (SEQ ID NO:95),-PSCK- (SEQ ID NO:101), -HTCK- (SEQ ID NO:98), -HTCK- (SEQ ID NO:98), or-PTCK- (SEQ ID NO:104) are contemplated by the present invention.

It is further contemplated that, in some embodiments, the anti-invasiveagents named above comprise the named amino acid sequence and additionalamino acids added to the amino terminus, the carboxyl terminus, or boththe amino and carboxyl termini (in the manner set forth above for thePHSRN (SEQ ID NO:1) containing peptides, e.g., PHSRNSIT (SEQ ID NO:5)).In one embodiment, the anti-invasive agent is up to five hundred aminoacids in length. It is also contemplated that, in some embodiments, theanti-invasive agents named above comprise a peptide with the aminoterminus blocked by standard methods to prevent digestion byexopeptidases, for example by acetylation; and the carboxyl terminusblocked by standard methods to prevent digestion by exopeptidases, forexample, by amidation.

In this regard, the present invention provides a method of treatingcancer comprising: a) providing: i) a subject having cancer, and ii) acomposition of matter comprising a peptide, peptide derivative, orpeptide mimetic which inhibits the tumor invasion-promoting activity ofa peptide comprising the amino acid sequence PHSRN (SEQ ID NO:1), and b)administering said composition to said subject. The present inventionfurther contemplates using antagonists before and/or after surgicalremoval of the primary tumor. In one embodiment, the method comprisesadministering a PHSRN (SEQ ID NO:1) antagonist as adjunct therapy withadditional chemotherapeutics.

While not limited to any mechanism, it is believed that theseanti-invasive chemotherapeutic agents antagonize the invasion-promotingactivity of the PHSRN (SEQ ID NO:1) sequence (e.g., of fibronectin) byblocking the binding of this sequence to its receptor on tumor cells.Again, while not limited to any mechanism, it is believed that the PHSRN(SEQ ID NO:1) sequence may promote invasion by acting to displace adivalent cation (Mg+2, Ca+2, or Mn+) in the α5β1 receptor on metastatictumor cells, and the above named chemotherapeutic anti-invasive agentsmight act to inhibit this invasion by chelating one or more of thesedivalent cations.

In another embodiment, the present invention contemplates anti-invasionantagonists to the IKVAV (SEQ ID NO:2) sequence of laminin.

2. Designing Mimetics

Compounds mimicking the necessary conformation for recognition anddocking to the receptor binding to the peptides of the present inventionare contemplated as within the scope of this invention. For example,mimetics of PHSRN (SEQ ID NO:1) and PHSRN-antagonists (SEQ ID NO:1) arecontemplated. A variety of designs for such mimetics are possible. Forexample, cyclic PHSRN (SEQ ID NO:1) and PHSCN (SEQ ID NO:86) containingpeptides, in which the necessary conformation for binding is stabilizedby nonpeptides, are specifically contemplated. U.S. Pat. No. 5,192,746to Lobl, et al., U.S. Pat. No. 5,169,862 to Burke, Jr., et al., U.S.Pat. No. 5,539,085 to Bischoff, et al., U.S. Pat. No. 5,576,423 toAversa, et al., U.S. Pat. No. 5,051,448 to Shashoua, and U.S. Pat. No.5,559,103 to Gaeta, et al., all hereby incorporated by reference,describe multiple methods for creating such compounds.

Synthesis of nonpeptide compounds that mimic peptide sequences is alsoknown in the art. Eldred, et al., (J. Med. Chem. 37:3882 (1994))describe nonpeptide antagonists that mimic the Arg-Gly-Asp sequence.Likewise, Ku, et al., (J. Med. Chem. 38:9 (1995)) give furtherelucidation of the synthesis of a series of such compounds. Suchnonpeptide compounds that mimic PHSRN (SEQ ID NO:1) andPHSRN-antagonists (SEQ ID NO:1) are specifically contemplated by thepresent invention.

The present invention also contemplates synthetic mimicking compoundsthat are multimeric compounds that repeat the relevant peptidesequences. In one embodiment of the present invention, it iscontemplated that the relevant peptide sequence is Pro-His-Ser-Arg-Asn(SEQ ID NO:1); in another embodiment, the relevant peptide sequence isPro-His-Ser-Cys-Asn (SEQ ID NO:86); in another embodiment, the relevantpeptide sequence is Ile-Lys-Val-Ala-Val (SEQ ID NO:2). As is known inthe art, peptides can be synthesized by linking an amino group to acarboxyl group that has been activated by reaction with a couplingagent, such as dicyclohexylcarbodiimide (DCC). The attack of a freeamino group on the activated carboxyl leads to the formation of apeptide bond and the release of dicyclohexylurea. It can be necessary toprotect potentially reactive groups other than the amino and carboxylgroups intended to react. For example, the α-amino group of thecomponent containing the activated carboxyl group can be blocked with atertbutyloxycarbonyl group. This protecting group can be subsequentlyremoved by exposing the peptide to dilute acid, which leaves peptidebonds intact. With this method, peptides can be readily synthesized by asolid phase method by adding amino acids stepwise to a growing peptidechain that is linked to an insoluble matrix, such as polystyrene beads.The carboxyl-terminal amino acid (with an amino protecting group) of thedesired peptide sequence is first anchored to the polystyrene beads. Theprotecting group of the amino acid is then removed. The next amino acid(with the protecting group) is added with the coupling agent. This isfollowed by a washing cycle. The cycle is repeated as necessary.

In one embodiment, the mimetics of the present invention are peptideshaving sequence homology to the above-described PHSRN (SEQ ID NO:1)sequences and PHSRN-antagonists (SEQ ID NO:1). One common methodologyfor evaluating sequence homology, and more importantly statisticallysignificant similarities, is to use a Monte Carlo analysis using analgorithm written by Lipman and Pearson to obtain a Z value. Accordingto this analysis, a Z value greater than 6 indicates probablesignificance, and a Z value greater than 10 is considered to bestatistically significant. W. R. Pearson and D. J. Lipman, Proc. Natl.Acad. Sci. (USA), 85:2444-2448 (1988); D. J. Lipman and W. R. Pearson,Science, 227:1435-1441 (1985). In the present invention, syntheticpolypeptides useful in tumor therapy and in blocking invasion are thosepeptides with statistically significant sequence homology and similarity(Z value of Lipman and Pearson algorithm in Monte Carlo analysisexceeding 6).

3. Antibody Inhibitors

The present invention contemplates all types of inhibitors of tumorinvasion for use in both the assays and for therapeutic use. In oneembodiment, the present invention contemplates antibody inhibitors. Theantibodies may be monoclonal or polyclonal, but polyclonal antibodiesare often more effective inhibitors. It is within the scope of thisinvention to include any second antibodies (monoclonal or polyclonal)directed to the first antibodies discussed above. Both the first andsecond antibodies may be used in the detection assays or a firstantibody may be used with a commercially available anti-immunoglobulinantibody. An antibody as contemplated herein includes any antibodyspecific to any region of a peptide involved in the induction of tumorcell invasion. For example, the present invention contemplatesantibodies reactive with PHSRN (SEQ ID NO:1) peptides (as well as therelated peptides set forth above).

Both polyclonal and monoclonal antibodies are obtainable by immunizationwith peptides, as well as with enzymes or proteins, and all types areutilizable for immunoassays. The methods of obtaining both types of seraare well known in the art. Polyclonal sera are less preferred but arerelatively easily prepared by injection of a suitable laboratory animalwith an effective amount of the purified enzyme or protein, or antigenicparts thereof, collecting serum from the animal, and isolating specificsera by any of the known immunoadsorbent techniques. Although antibodiesproduced by this method are utilizable in virtually any type ofimmunoassay, they are generally less favored because of the potentialheterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularlypreferred because of the ability to produce them in large quantities andthe homogeneity of the product. The preparation of hybridoma cell linesfor monoclonal antibody production derived by fusing an immortal cellline and lymphocytes sensitized against the immunogenic preparation canbe done by techniques which are well known to those who are skilled inthe art. (See, for example Douillard and Hoffman, Basic Facts aboutHybridomas, in Compendium of Immunology Vol II, ed. by Schwartz, 1981;Kohler and Milstein, Nature 256: 495-499, 1975; European Journal ofImmunology 6: 511-519, 1976).

Unlike preparation of polyclonal sera, the choice of animal is dependenton the availability of appropriate immortal lines capable of fusing withlymphocytes. Mouse and rat have been the animals of choice in hybridomatechnology and are preferably used. Humans can also be utilized assources for sensitized lymphocytes if appropriate immortalized human (ornonhuman) cell lines are available. For the purpose of the presentinvention, the animal of choice may be injected with an antigenicamount, for example, from about 0.1 mg to about 20 mg of the enzyme orprotein or antigenic parts thereof. Usually the injecting material isemulsified in Freund's complete, adjuvant. Boosting injections may alsobe required. The detection of antibody production can be carried out bytesting the antisera with appropriately labelled antigen. Lymphocytescan be obtained by removing the spleen of lymph nodes of sensitizedanimals in a sterile fashion and carrying out fusion. Alternatively,lymphocytes can be stimulated or immunized in vitro, as described, forexample, in Reading, Journal of Immunological Methods 53: 261-291, 1982.

A number of cell lines suitable for fusion have been developed and thechoice of any particular line for hybridization protocols is directed byany one of a number of criteria such as speed, uniformity of growthcharacteristics, deficiency of its metabolism for a component of thegrowth medium, and potential for good fusion frequency.

Intraspecies hybrids, particularly between like strains, work betterthan interspecies fusions. Several cell lines are available, includingmutants selected for the loss of ability to secrete myelomaimmunoglobulin.

Cell fusion can be induced either by virus, such as Epstein-Barr orSendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is themost efficacious agent for the fusion of mammalian somatic cells. PEGitself may be toxic for cells and various concentrations should betested for effects on viability before attempting fusion. The molecularweight range of PEG may be varied from 1000 to 6000. It gives bestresults when diluted to from about 20% to about 70% (w/w) in saline orserum-free medium. Exposure to PEG at 37° C. for about 30 seconds ispreferred in the present case, utilizing murine cells. Extremes oftemperature (i.e., about 45° C.) are avoided, and preincubation of eachcomponent of the fusion system at 37° C. prior to fusion can be useful.The ratio between lymphocytes and malignant cells is optimized to avoidcell fusion among spleen cells and a range of from about 1:1 to about1:10 is commonly used.

The successfully fused cells can be separated from the myeloma line byany technique known by the art. The most common and preferred method isto choose a malignant line which is Hypoxthanine Guanine PhosphoribosylTransferase (HGPRT) deficient, which will not grow in anaminopterin-containing medium used to allow only growth of hybrids andwhich is generally composed of hypoxthanine 1×10-4M, aminopterin1×10-5M, and thymidine 3×10-5M, commonly known as the HAT medium. Thefusion mixture can be grown in the HAT-containing culture mediumimmediately after the fusion 24 hours later. The feeding schedulesusually entail maintenance in HAT medium for two weeks and then feedingwith either regular culture medium or hypoxthanine, thymidine-containingmedium.

The growing colonies are then tested for the presence of antibodies thatrecognize the antigenic preparation. Detection of hybridoma antibodiescan be performed using an assay where the antigen is bound to a solidsupport and allowed to react to hybridoma supernatants containingputative antibodies. The presence of antibodies may be detected by“sandwich” techniques using a variety of indicators. Most of the commonmethods are sufficiently sensitive for use in the range of antibodyconcentrations secreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth inselected medium. Cloning can be preformed by cell limiting dilution influid phase or by directly selecting single cells growing in semi-solidagarose. For limiting dilution, cell suspensions are diluted serially toyield a statistical probability of having only one cell per well. Forthe agarose technique, hybrids are seeded in a semi-solid upper layer,over a lower layer containing feeder cells. The colonies from the upperlayer may be picked up and eventually transferred to wells.

Antibody-secreting hybrids can be grown in various tissue cultureflasks, yielding supernatants with variable concentrations ofantibodies. In order to obtain higher concentrations, hybrids may betransferred into animals to obtain inflammatory ascites.Antibody-containing ascites can be harvested 8-12 days afterintraperitoneal injection. The ascites contain a higher concentration ofantibodies but include both monoclonals and immunoglobulins from theinflammatory ascites. Antibody purification may then be achieved by, forexample, affinity chromatography.

A wide range of immunoassay techniques are available for evaluating theantibodies of the present invention as can be seen by reference to U.S.Pat. Nos. 4,016,043; 4,424,279 and 4,018,653, hereby incorporated byreference. This, of course, includes both single-site and two-site, or“sandwich”, assays of the non-competitive types, as well as in thetraditional competitive binding assays.

4. Administering Chemotherapeutics

It is contemplated that the antagonists of the present invention beadministered systemically or locally to inhibit tumor cell invasion incancer patients with locally advanced or metastatic cancers. They can beadministered intravenously, intrathecally, intraperitoneally as well asorally. PHSRN (SEQ ID NO:1) antagonists (e.g., the PHSCN (SEQ ID NO:86)peptide), can be administered alone or in combination withanti-proliferative drugs in a neoadjuvant setting to reduce themetastatic load in the patient prior to surgery; or they can beadministered after surgery. Since PHSRN (SEQ ID NO:1) antagonists maydepress wound healing (because the PHSRN (SEQ ID NO:1) sequence alsoelicits fibroblast invasion as described below), it may be necessary touse PHSRN (SEQ ID NO:1) antagonists some time after surgery to removethe tumor.

Since few cells in the body must invade in order to function, PHSRN (SEQID NO:1) antagonists administered systemically are not likely to causethe debilitating side effects of cytotoxic chemotherapeutic agents.However, since they suppress invasion, they are likely to cause someimmunodepression. Even so, at the appropriate dosage, PSHRN (SEQ IDNO:1) antagonists may be administered prophylactically. In any case, itis contemplated that they may be administered in combination withcytotoxic agents. The simultaneous selection against the two fatalattributes of metastatic cells, unrestricted proliferation and invasion,is contemplated as a very powerful therapeutic strategy.

Where combinations are contemplated, it is not intended that the presentinvention be limited by the particular nature of the combination. Thepresent invention contemplates combinations as simple mixtures as wellas chemical hybrids. An example of the latter is where the antagonist iscovalently linked to a targeting carrier or to an active pharmaceutical.Covalent binding can be accomplished by any one of many commerciallyavailable crosslinking compounds.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided together with physiologically tolerableliquid, gel or solid carriers, diluents, adjuvants and excipients.

These therapeutic preparations can be administered to mammals forveterinary use, such as with domestic animals, and clinical use inhumans in a manner similar to other therapeutic agents. In general, thedosage required for therapeutic efficacy will vary according to the typeof use and mode of administration, as well as the particularizedrequirements of individual hosts.

Such compositions are typically prepared as liquid solutions orsuspensions, or in solid forms. Oral formulations for cancer usuallywill include such normally employed additives such as binders, fillers,carriers, preservatives, stabilizing agents, emulsifiers, buffers andexcipients as, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, magnesiumcarbonate, and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations,or powders, and typically contain 1%-95% of active ingredient,preferably 2%-70%.

The compositions are also prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared.

The antagonists of the present invention are often mixed with diluentsor excipients which are physiological tolerable and compatible. Suitablediluents and excipients are, for example, water, saline, dextrose,glycerol, or the like, and combinations thereof. In addition, if desiredthe compositions may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, stabilizing or pH buffering agents.

Additional formulations which are suitable for other modes ofadministration, such as topical administration, include salves,tinctures, creams, lotions, and, in some cases, suppositories. Forsalves and creams, traditional binders, carriers and excipients mayinclude, for example, polyalkylene glycols or triglycerides.

Anti-Thrombotics

In addition to using the PHSRN (SEQ ID NO:1) antagonists described aboveas anti-invasion chemotherapeutics, it is also contemplated that theseantagonists be used as anti-thrombotics. This use of the PHSRN (SEQ IDNO:1) antagonists described above is based on the discovery that PHSCN(SEQ ID NO:86) peptide-treated blood appears in vivo to clot veryslowly.

A number of anti-thrombotic agents are currently known which inhibitclot formation by preventing platelet integrins from binding fibrinogenor fibronectin. These anti-thrombotics, however, rely on competitiveinhibition to prevent platelet integrins from binding to fibrinogen orfibronectin. In this manner, large doses of these agents are required toachieve the desired anti-thrombotic affect.

The present invention contemplates a more effective approach usingPHSRN-antagonists (SEQ ID NO:1) such as PHSCN (SEQ ID NO:86). While theprecise mechanism need not be known to practice the invention it hasbeen shown that the platelet integrin, αIIbβ3, also binds the PHSRN (SEQID NO:1) sequence of plasma fibronectin. Thus, instead of utilizingcompetitive inhibition, the PHSRN-antagonists (SEQ ID NO:1) may directlyinhibit platelet integrins from binding fibronectin and aggregating.Specifically, the PHSCN (SEQ ID NO:86) peptide, or otherPHSRN-antagonists (SEQ ID NO:1), may directly inhibit early stages inclot formation by binding to the αIIbβ3 receptors on platelets. Thisprevents platelet integrins from binding fibronectin, a necessary partof platelet aggregation, thus inhibiting an integral step in the bloodclotting cascade. In this manner, a comparatively small dose of thePHSCN (SEQ ID NO:86) peptide, or other PHSRN (SEQ ID NO:1) antagonist,is contemplated as effective anti-thrombotic agents.

Wound Healing

As noted above, it is contemplated that PHSRN (SEQ ID NO:1) antagonistsmay depress wound healing. This expectation is based on the discoverythat PHSRN-containing (SEQ ID NO:1) peptides promote wound healing.

In this regard, it should be noted that the therapy of wounds,particularly those which are made difficult to heal by disease, has beenattempted with a variety of purified growth factors or cytokines becausethese molecules can induce cellular proliferation or increase themotility of cells in wounds. Thus, if presented in the correct form andlocation at the right time, growth factors may greatly accelerate orenhance the healing of wounds by stimulating the growth of new tissue.Given the complexity and clinical variability of wounds, an obviousdifficulty with the application of specific, purified growth factors orcytokines to wounded tissue, alone or in combination, is that theirforms or specific distributions in the wound may not support theirnormal activities. Instead, the effectiveness of growth factors andcytokines in promoting the healing of wounded tissue may depend on theirsecretion by fibroblasts or macrophages.

The present invention contemplates a more effective approach; thisapproach involves methods that stimulate the invasion of the wound bythe cells which synthesize the growth factors and cytokines active instimulating wound repair, especially monocytes, macrophages, andfibroblasts. This strategy allows the cells in their normal in vivosetting to secrete the active factors. This approach has a number ofadvantages: (1) the temporal and spatial distributions of the factorsare likely to be optimal because the normally active cells in theircorrect settings are secreting them; (2) all the appropriate factors arelikely to be present in their active forms, irrespective of whether theyhave been identified or cloned; (3) the sequential effects of thefactors in recruiting subsequent waves of cells involved in the healingprocess to the wound site are likely to be enhanced by the presence ofmore initiating cells in the wound.

The present invention is based on the discovery that the pure PHSRN (SEQID NO:1) peptide or purified plasma fibronectin fragments containing it,and lacking the α4β1 integrin binding site in the IIICS region, aresufficient to stimulate fibroblast invasion of basement membranes invitro in the presence of serum or under serum-free conditions, whileintact plasma fibronectin fails to stimulate fibroblast invasion. PurePHSRN (SEQ ID NO:1) peptide has also been shown to stimulatekeratinocyte invasion of serum-free SU-ECM. Since, during woundreepithelialization, keratinocytes migrate through the connective tissueof the provisional matrix to “wall off” portions of the wound, as wellas through the adjacent stroma, it is not surprising that they are alsostimulated to migrate through the matrix of SU-ECM invasion substratesby the PHSRN (SEQ ID NO:1) sequence. This suggests that this peptide, orproteinase-resistant forms of it, may have similar effects onfibroblasts, keratinocytes, and monocytes/macrophages in vivo.Recruitment of fibroblasts or monocytes/macrophages whose paracrine,regulatory effects on a variety of neighboring cells are required forthe early stages of wound healing is contemplated as a highly efficientand effective way to stimulate the cascade of regulatory interactionsinvolved in wound healing because these cells will secrete the activefactors or cytokines in the correct temporal sequences and spatiallocations to ensure their optimal activities. Because it efficientlyinduces keratinocyte migration through the extracellular matrix invitro, the PHSRN (SEQ ID NO:1) peptide is also likely to stimulate woundreepithelialization directly. The use of the PHSRN (SEQ ID NO:1) peptideor structurally related molecules according to the present invention isto stimulate the entry of cells such as fibroblasts andmonocyte/macrophages into the provisional matrix of a wound, so that theentering cells themselves secrete the factors and cytokines active ininducing or potentiating wound healing. The use of the PHSRN (SEQ IDNO:1) peptide or structurally related molecules is also intended tostimulate wound reepithelialization directly by inducing keratinocytemigration through the extracellular matrix.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); mM(millimolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); g (grams); mg (milligrams); μg(micrograms); L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); mAb (monoclonal antibody); MW (molecular weight);PBS (phophate buffered saline); U (units); d (days).

Example 1 Production of Fibronectin-Free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by affinity chromatography overGelatin-Sepharose (a technique which can be used to remove plasmafibronectin from fetal calf serum).

The Gelatin-Sepharose beads were obtained from Pharmacia (Catalog#17-0956-01). Two Kontes columns were set up with about 2 mls ofGelatin-Sepharose beads at 4 C to prevent gelling of the Matrigel. Thecolumns were then rinsed with about 10 column volumes of PBS to removethe preservative from the beads. The columns were drained to the top ofthe beads; then Matrigel was carefully added to the column. Once theMatrigel had entered the column, PBS was added to the top of the column.The Matrigel which was passed over the first column was collected andpassed over the second column. The fibronectin-depleted Matrigelcollected from the second column was plated on 48-well plates (150μl/well), sterilized under a UV light for 10 minutes and incubated at 37C overnight. The Matrigel treated in this manner failed to form a gel at37 C.

Example 2 Production of Fibronectin-Free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by successive panning on gelatin.Eight wells of 24-well plate were coated with a 2% gelatin solution (thegelatin was obtained from Becton Dickinson Labware, Catalog #11868). Thewells were filled with the gelatin solution which had been heated to 50C and incubated for 3 minutes. Then the solution was removed and thewells were allowed to air dry. Following drying, the wells werethoroughly rinsed with ddH2O followed by two rinses with PBS. The plateswere again allowed to dry; thereafter they were stored at −20 C untiluse. Matrigel was thawed on ice and then added to one of the wells of agelatin-coated plate (between 800 μl and 1 ml of Matrigel was added to awell of a 24-well plate). The plate was placed in a bucket of ice in a 4C room on an orbital shaker where the Matrigel was incubated in the wellfor two hours (although overnight incubation can be used). Following theincubation, the Matrigel was moved from the first well to a second welland then incubated for two hours under the same conditions. This processwas repeated until the Matrigel had been incubated on all eight wells ofthe gelatin-coated plate.

Following the depletion of the Matrigel, it was collected in Eppendorftubes. It was then plated on a 48-well plate 150 μl/well), sterilizedunder a UV light for 10 minutes and incubated at 37 C overnight. TheMatrigel formed as gel and the following day, cells were added to eachwell.

Example 3 Production of Fibronectin-Free Substrates

This example describes a purification approach for removal of plasmafibronectin (and/or cellular fibronectin) from a substrate (Matrigel).In this example, removal was attempted by gelatin panning followed byantibody panning.

Anti-fibronectin antibody-coated wells: Wells of a 24-well plate werecoated with an anti-fibronectin antibody. A mouse monoclonal antibody tohuman fibronectin was obtained from Oncogene Science (Catalog #CP13).Each well was incubated with 1 ml of antibody at a concentration of 30μl/ml for 2 hours at room temperature. Each well was then incubated witha solution of 3% BSA in PBS for 2 hours at room temperature. Followingthe two incubation periods, the wells were thoroughly washed with PBSand stored at −20 C until use.

Depleting Matrigel of Fibronectin: Matrigel was panned over eightgelatin-coated wells (as described above in Example 2) to remove most ofthe fibronectin and its fragments. Thereafter, the Matrigel was placedin the antibody-coated wells to remove any remaining fragments offibronectin which contain the cell-binding domain but not thegelatin-binding domain. The Matrigel was incubated in an ice bucket onan orbital shaker at 4 C for 2 hours. Once the Matrigel was depleted, itwas collected in Eppendorf tubes. The firbonectin-depleted Matrigel wasplated on a 48-well plate (150 μl/well), sterilized under a UV light for10 minutes and incubated at 37 C overnight. The Matrigel formed a geland the following day, cells were added to the wells.

Example 4 Inducing Invasive Behavior of Tumor Cells

In this example, the role of plasma fibronectin in inducing the invasivebehaviors of metastatic breast and prostate cancer cells isdemonstrated. Human breast carcinoma cell lines SUM 52 PE and SUM 44 PEwere originally cultured from the pleural effusions of patients withmetastatic breast cancer; and SUM 102 was cultured from a primary,microinvasive breast carcinoma (Ethier, S. P., Mahack, M. L., Gullick,W. J., Frank, T. S., and Weber, B. L. Differential isolation of normalluminal mammary epithelial cells and breast cancer cells from primaryand metastatic sites using selective media. Cancer Res. 53: 627-635).The DU 145 metastatic human prostate cancer cell line was originallycultured from a brain metastasis (Stone, K. R., Mickey, D. D., Wunderli,H., Mickey, G. H., Paulsen, D. F. (1978) Isolation of a human prostatecarcinoma cell line (DU 145), Int. J. Cancer 21: 274-281. These cellsexpress α3μ1 which has been shown to repress metalloproteinasetranscription upon binding the connecting segment of plasma Fn. Thesecell lines can all be cultured under serum-free conditions; thus theyare ideal for use in serum-free invasion assays on SU-ECM.

Adult Strongylocentrotus purpuratus sea urchins were obtained fromPacific BioMarine, and their embryos were cultured to the early pluteusstage in artificial sea water at 15° C. SU-ECM were prepared from themby treatment with nonionic detergent and strerilized by dilution in theappropriate media.

Cells were harvested by rinsing in Hanks' balanced salt solution,followed by brief treatment with 0.25% trypsin, 0.02% EDTA, andpelleting and resuspension in the appropriate medium with or without 5%FCS at a density of about 50,000 cells per ml. When appropriate,purified bovine plasma fibronectin (Sigma), purified 120 kDachymotryptic fragment (Gibco BRL), PHSRN (SEQ ID NO:1) or PHSCN (SEQ IDNO:86) peptides (synthesized at the Biomedical Research Core Facilitiesof the University of Michigan), or GRGDSP (SEQ ID NO:83) or GRGESP (SEQID NO:84) peptides (Gibco BRL) were added to the resuspended cells priorto placement of the cells on SU-ECM. In each well of a plate used for aninvasion assay, SU-ECM were placed in 0.5 ml of the appropriate medium,and 0.5 ml of the resuspended cells dropped on their exterior surfaces.Invasion assays were incubated 1 to 16 hours prior to assay. If somecircumstances, invasion assays were fixed in phosphate-buffered saline(PBS) with 2% formaldehyde for 5 minutes at room temperature, thenrinsed into PBS.

Invasion assays were coded and scored blindly by microscopic examinationunder phase contrast at 200- and 400-fold magnification. Each cellcontacting an SU-ECM was scored for its position relative to theexterior or interior surfaces. A cell was judged to have invaded if itwas located on an interior surface below the focal plane passing throughthe upper surface of the SU-ECM, but above the focal plane passingthrough its lower surface. The minimum viability of the cells in eachassay was always ascertained at the time of assay by determining thefraction of spread, adherent cells on the bottom of each well scored.

An invasion frequency is defined as the fraction of cells in contactwith basement membranes which were located in their interiors at thetime of assay. Thus, an invasion frequency of 1 denotes invasion by 100%of the cells in contact with basement membranes. Invasion frequencieswere determined multiple times for each cell type assayed. For each typeof cell assayed the mean and standard deviation of the invasionfrequencies were calculated.

The invasion-inducing sequences of plasma fibronectin were mapped to apeptide sequence 5 amino acids long, the PHSRN (SEQ ID NO:1) peptide,for both metastatic breast and prostate cancer cells. Since the PHSRN(SEQ ID NO:1) sequence is present in plasma fibronectin, a significantcomponent of serum, eliciting the regulatory role of this sequence wasonly possible because of the availability of a serum-free in vitroinvasion substrate. It should be noted that neonatal, human fibroblastsare also induced with the PHSRN (SEQ ID NO:1) peptide to invadeserum-free SU-ECM. Although fibroblasts do not invade SU-ECM in thepresence of serum, the 120 kDa fragment of plasma fibronectin containingthe PHSRN (SEQ ID NO:1) sequence can induce fibroblast invasion equallywell in the presence of serum or in its absence.

When taken together, the results of experiments showing that the PHSRN(SEQ ID NO:1) sequence of plasma fibronectin induces the invasivebehaviors of both metastatic breast and prostate cancer cells, as wellas that of normal fibroblasts suggest the intriguing possibility thatthe invasive behavior associated with tumor cell metastasis may resultfrom defects in the regulation of the normal invasive behaviorsassociated with wound healing.

Example 5 Testing Tumor Cells on Fibronectin-Depleted Substrates

This example describes an approach to test cancer cells in vitro onsubstrates with and without invasion-inducing agents. The depletedpreparation of Matrigel (see Example 2, above) and untreated Matrigelwere used to test DU-145 metastatic prostate cancer cells. When platedon the depleted medium, the cancer cells failed to invade the matrix(see FIG. 2). Indeed, it was evident that these cells were sitting onthe surface of the depleted Matrigel because the Matrigel surface wasslightly tilted; this was visible through the microscope as a gradualprogressive, uniform change in the focal plane for the monolayer ofDU-145 cells.

The addition of 0.5 μl/ml of the PHSRN (SEQ ID NO:1) peptide to thedepleted Matrigel was sufficient to restore the full DU-145 invasiveness(see FIG. 3). Clearly, gelatin panning removes fibronectin such thatcancer cells are unable to invade. Since the addition of PHSRN (SEQ IDNO:1) peptide in solution fully restores the DU-145 invasive phenotype,blocking the effect of PHSRN (SEQ ID NO:1) is an effective strategy fortherapeutic intervention in tumor cell invasion and metastasis.

Example 6 Improving Gelatin Depletion as Measured by FibroblastInvasiveness

In this example, normal, neonatal fibroblasts were tested on thedepleted Matrigel material prepared according to Example 3 above (i.e.,antibody depletion). As shown in FIG. 4, panning with an antibody aftergelatin depletion improved the method for removal, as measured by thereduced invasiveness of fibroblasts. On the other hand, invasiveness ofthe fibroblasts could be induced by the addition of the PHSRN (SEQ IDNO:1) peptide.

The success of antibody panning suggests the feasibility of removingother components by the antibody panning methods. Other serumcomponents, such as thrombospondin, growth factors and cytokines arecontemplated by the present invention for removal by the appropriate(commercially available) antibody.

Example 7 Conjugation of PHSRN-Containing (SEQ ID NO:1) Peptides

In this example, the preparation of a peptide conjugate is described.The synthetic peptide NH₂-PHSRNC (SEQ ID NO:82) can be preparedcommercially (e.g., Multiple Peptide Systems, San Diego, Calif.). Thecysteine is added to facilitate conjugation to other proteins.

In order to prepare a protein for conjugation (e.g., BSA), it isdissolved in buffer (e.g., 0.01 M NaPO₄, pH 7.0) to a finalconcentration of approximately 20 mg/ml. At the same timen-maleimidobenzoyl-N-hydroxysuccinimide ester (“MBS” available fromPierce) is dissolved in N,N-dimethyl formamide to a concentration of 5mg/ml. The MBS solution, 0.51 ml, is added to 3.25 ml of the proteinsolution and incubated for 30 minutes at room temperature with stirringevery 5 minutes. The MBS-activated protein is then purified bychromatography on a Bio-Gel P-10 column (Bio-Rad; 40 ml bed volume)equilibrated with 50 mM NaPO₄, pH 7.0 buffer. Peak fractions are pooled(6.0 ml).

The above-described cysteine-modified peptide (20 mg) is added to theactivated protein mixture, stirred until the peptide is dissolved andincubated 3 hours at room temperature. Within 20 minutes, the reactionmixture becomes cloudy and precipitates form. After 3 hours, thereaction mixture is centrifuged at 10,000×g for 10 min and thesupernatant analyzed for protein content. The conjugate precipitate iswashed three times with PBS and stored at 4° C.

From the above, it should be clear that the present invention provides amethod of testing a wide variety of tumor types, and in particularidentifying invasive tumors. With a means of identifying such tumors(now provided by the present invention) and distinguishing such tumorsfrom non-invasive cancer, the physician is able to change and/oroptimize therapy. Importantly, the antagonists of the present invention(and other drugs developed by use of the screening assay of the presentinvention) will provide treatment directed an invasive cells (andtherefore associated with minimal host toxicity).

Example 8 Inhibiting Invasion of Human Breast Cancer Cells

In this example, the role of the PHSCN (SEQ ID NO:86) peptide ininhibiting the invasive behavior of metastatic breast cancer cells isdemonstrated. The method of Example 4 is employed, with the addition ofvarying concentrations of the PHSCN (SEQ ID NO:86) peptide.

Example 4 indicates that SUM-52 cells (in medium with 5% fecal calfserum) are induced to invade the SU-ECM substrate in the presence ofserum fibronectin or just the PHSRN (SEQ ID NO:1) sequence offibronectin. Thus, the procedure in Example 4 provides a method fordetermining the inhibitory potential of the PHSCN (SEQ ID NO:86) peptideby comparing the number of cell invasions in the presence of the PHSCN(SEQ ID NO:86) peptide, with the number of cell invasions in the absenceof the PHSCN (SEQ ID NO:86) peptide.

The results of adding varying concentrations of the PHSCN (SEQ ID NO:86)peptide to serum-induced metastatic SUM-52 PE breast cancer cells arepresented in FIG. 5A. The logs of the PHSCN (SEQ ID NO:86) peptideconcentrations in ng per ml are plotted on the X axis. The percentagesof invaded SUM 52 PE cells relative to the percentage invaded in theabsence of the PHSCN (SEQ ID NO:86) peptide are plotted on the Y axis.Mean invasion percentages are shown with their first standarddeviations. Clearly, the PHSCN (SEQ ID NO:86) peptide exhibits asubstantial inhibitory affect on these cells, even at relatively lowconcentrations. The PHSCN (SEQ ID NO:86) peptide's inhibitory affect isfurther demonstrated by the fact that relatively high concentrationscause complete inhibition.

The results of adding varying concentrations of the PHSCN (SEQ ID NO:86)peptide to PHSRN-induced (SEQ ID NO:1) metastatic SUM-52 PE breastcancer cells (in serum free medium) and normal human mammary epithelialcells (in 10% FCS), are presented in FIG. 5B. All invasion assays werecarried out in 100 ng per ml of the PHSRN (SEQ ID NO:1) peptide toinduce invasion. Again, the PHSCN (SEQ ID NO:86) peptide exhibits asubstantial inhibitory affect on both cell lines at low concentrations,and almost complete inhibition at higher concentrations.

This example demonstrates the PHSCN (SEQ ID NO:86) peptide is aneffective inhibitor of human breast cancer cell invasion. In thismanner, the PHSCN (SEQ ID NO:86) peptide, or related sequences, arelikely to provide effective therapy for human breast cancer bypreventing the lethal affects of tumor cell metastasis.

Example 9

Inhibiting Invasion of Human Prostate Cancer Cells

In this example, the role of the PHSCN (SEQ ID NO:86) peptide ininhibiting the invasive behavior of metastatic prostate cancer cells isdemonstrated. The method of Example 4 is employed, with the addition ofvarying concentrations of the PHSCN (SEQ ID NO:86) peptide.

Example 4 indicates that DU-145 cells are induced to invade the SU-ECMsubstrate in the presence of serum fibronectin or just the PHSRN (SEQ IDNO:1) sequence of fibronectin. Thus, the procedure in Example 4 providesa method for determining the inhibitory potential of the PHSCN (SEQ IDNO:86) peptide by comparing the number of cell invasions in the presenceof the PHSCN (SEQ ID NO:86) peptide, with the number of cell invasionsin the absence of the PHSCN (SEQ ID NO:86) peptide.

The results of adding varying concentrations of the PHSCN (SEQ ID NO:86)peptide to serum-induced invasion of metastatic DU-145 prostate cancercells (in 10% serum) are presented in FIG. 6A. The logs of the PHSCN(SEQ ID NO:86) concentrations are plotted on the X axis. The percentagesof invaded DU-145 cells relative to the percentage invaded in theabsence of the PHSCN (SEQ ID NO:86) peptide are plotted on the Y axis.Mean invasion percentages are shown with their first standarddeviations. Clearly, the PHSCN (SEQ ID NO:86) peptide exhibits asubstantial inhibitory affect on these cells, even at relatively lowconcentrations. The PHSCN (SEQ ID NO:86) peptide's inhibitory affect isfurther demonstrated by the fact that relatively high concentrationscause complete inhibition.

The results of adding varying concentrations of the PHSCN (SEQ ID NO:86)peptide to PHSRN-induced (SEQ ID NO:1) metastatic DU-145 prostate cancercells (in serum-free medium) and normal human prostate epithelial cells(in 10% FCS), are presented in FIG. 6B. All invasion assays were carriedout in 100 ng per ml of the PHSRN (SEQ ID NO:1) peptide to induceinvasion. Again, the results show that the PHSCN (SEQ ID NO:86) peptideexhibits a substantial inhibitory affect on both cell lines at lowconcentrations, and almost complete inhibition at higher concentrations.

This example demonstrates the PHSCN (SEQ ID NO:86) peptide is aneffective inhibitor of human prostate cancer cell invasion. In thismanner, the PHSCN (SEQ ID NO:86) peptide may provide an effectivetherapy for human prostate cancer by preventing the lethal affects oftumor cell metastasis.

Example 10 Inhibiting Invasion of Rat Prostate Cancer Cells

In this example, the role of the PHSCN (SEQ ID NO:86) peptide ininhibiting the invasive behavior of rat metastatic prostate carcinomaMatLyLu (MLL) cells is demonstrated (see Example 4 for the generalprocedure employed). The result of adding 1 microgram per ml of thePHSCN (SEQ ID NO:86) peptide to serum-induced MLL cells causes completeinhibition of invasion (see FIG. 7A).

The result of adding a varying concentration of the PHSCN (SEQ ID NO:86)peptide to PHSRN-induced (SEQ ID NO:1) MLL cells in serum free media isshown in FIG. 7B, where 100 ng per ml of PHSRN (SEQ ID NO:1) was used toinduce invasion. FIG. 7B indicates that the PHSCN (SEQ ID NO:86) peptideexhibits a substantial inhibitory affect even at low concentrations, andalmost complete inhibition at higher concentrations. This exampledemonstrates invasion of rat prostate cancer cells is inhibited in thesame manner as human breast cancer cells (see Example 8) and humanprostate cancer cells (see Example 9).

Example 11 Inhibiting Invasion of Rat Prostate Cancer Cells

In this example, the role of a homo-cysteine containing peptide (i.e.,PHS(hC)N) (SEQ ID NO:85) in inhibiting the invasive behavior of ratmetastatic prostate carcinoma MatLyLu (MLL) cells is demonstrated. Theprocedure described in Example 10, was employed using SU-ECM substratesin 10% FCS and PHS(hC)N (SEQ ID NO:85) instead of PHSCN (SEQ ID NO:86).The result of adding varying concentrations of the PHS(hC)N (SEQ IDNO:85) peptide to serum-induced MLL cells indicates this peptide alsohas an inhibitory affect on cell invasion (see FIG. 8). As with thePHSCN (SEQ ID NO:86) peptide, the PHS(hC)N (SEQ ID NO:85) peptidesubstantially inhibits invasion at lower concentrations, and completelyinhibits invasion at higher concentrations. This example demonstratesthat the PHS(hC)N (SEQ ID NO:85) peptide has a similar inhibitory affectas the PHSCN (SEQ ID NO:86) peptide.

Example 12 Inhibiting Growth and Metastasis of Prostate Cancer Tumors InVivo

In this example, the role of the PHSCN (SEQ ID NO:86) peptide ininhibiting the growth and metastasis of prostate cancer tumors in vivois demonstrated. In the first part of this example, four Copenhagen ratswere injected with 500,000 MatLyLu (MLL) cells subcutaneously in thethigh. Two of these rats also received 1 mg of the PHSCN (SEQ ID NO:86)peptide along with the injected MLL cells, and thereafter received 1 mgof the PHSCN (SEQ ID NO:86) peptide injected in their tail vein threetime per week for two weeks. The other two injected rats were leftuntreated. Tumor sizes were measured with calipers on day 14, and thetumors in the untreated rats were removed. The results depicted in FIG.9A, clearly demonstrate that the PHSCN (SEQ ID NO:86) peptidesignificantly slows the growth of injected MLL tumors in rats. It ispossible that the ability of the PHSCN (SEQ ID NO:86) peptide to slowtumor growth is due to its inhibition of tumor invasion by normalendothelial cells, an anti-angiogenic effect.

Two weeks after the size of the tumors were measured, the rats weresacrificed and the mean number of lung metastases was determined at10-fold magnification. The mean number of lung metastases in theuntreated mice (MLL only) was nearly 35 in spite of the fact that theinitial prostate tumors had been removed when their size was measured.The mean number of lung metastases in the treated mice (MLL+PHSCN (SEQID NO:86)) was less than 5, even though the initial prostate tumors werenever removed because they were too small. This striking difference inmean number of metastases, depicted in FIG. 9B, indicates that the PHSCN(SEQ ID NO:86) peptide significantly inhibits tumor cell metastasis inrats. In this manner, the PHSCN (SEQ ID NO:86) peptide provideseffective in vivo therapy for cancer by preventing the lethal effects oftumor cell growth and metastasis.

Example 13 Inhibiting Growth and Metastasis of Prostate Cancer In Vivo

In this example, as in Example 12, the role of the PHSCN (SEQ ID NO:86)peptide in inhibiting the growth and metastasis of prostate cancertumors in vivo is demonstrated. In the first part of this example, 20Copenhagen rats were injected with 500,000 MatLyuLu (MLL) cellssubcutaneously in the thigh. To more closely approximate a real clinicalsituation, PHSCN (SEQ ID NO:86) peptide treatment of 10 of these ratswas initiated after 24 hours, instead of immediately. The 10 treatedrats (MLL/PHSCN (SEQ ID NO:86)) received a total of 5 i.v. injections of1 mg of the PHSCN (SEQ ID NO:86) peptide through the tail vein over twoweeks. Tumor sizes were measured with calipers on day 14, and the tumorsin the untreated rats were removed. Since the injected tumors in theMLL/PHSCN (SEQ ID NO:86) rats were still small, they were retained inthe rats for another 7 to 9 days following the last PHSCN (SEQ ID NO:86)injection. At this time, their sizes were all greater than 2 cm, andthey were also removed. The result of the first part of this example,depicted in FIG. 10A, clearly indicates that the PHSCN (SEQ ID NO:86)peptide, even when administered after the tumor cells have “seeded”,substantially slows the growth of rat prostate cancer tumors.

The dramatic growth-inhibitory effect of the PHSCN (SEQ ID NO:86)peptide on MLL tumors may be due to their inhibition of the invasion ofhost endothelial cells into the tumor. Host endothelial cell invasionmay be induced by the secretion of large amounts of proteinases from thetumors, and the resulting fragmentation of host plasma fibronectin.Fibronectin fragments have been shown to stimulate themigratory/invasive behaviors of normal mesenchymal and endothelialcells. This angiogenic process is believed to occur during normal woundhealing. Thus, the ability of metastatic cells to be constitutivelyinduced by intact plasma fibronectin to express proteinases and invademay play a central role both in tumor cell invasion and in tumor growth.In this manner, the PHSCN (SEQ ID NO:86) peptide is an effectivechemotherapeutic to prevent the growth of tumors in vivo.

In the second part of this example, the MLL/PHSCN (SEQ ID NO:86) ratsreceived 2 more i.v. doses of the PHSCN (SEQ ID NO:86) peptide prior tosacrifice. Ten days after the sizes of the injected primary tumors weredetermined, all the rats in the two groups (MLL only and MLL/PHSCN (SEQID NO:86)) were sacrificed, and the number of lung metastases wasdetermined at 7.5-fold magnification. As can be seen in FIG. 10B, thereis a significant reduction in the mean numbers of lung metastases in therats which received PHSCN (SEQ ID NO:86) treatment as compared to theuntreated rats.

The 20 rats described in parts one and two of this example were alsoexamined for metastatic tissues in their lymphatic systems. All of thesemetastases were dissected and weighed. FIG. 10C plots the mean masses ofintraperitoneal metastases (grams) for the two groups of 10 rats. As isclearly demonstrated, there is a significant reduction in the meanmasses of lymphatic metastases in the rats which received PHSCN (SEQ IDNO:86) peptide treatment, as compared to the untreated rats. This may bedue to the anti-angiogenic effect of the PHSCN (SEQ ID NO:86) peptide,as described in part one of this example. In this manner, the PHSCN (SEQID NO:86) peptide maybe an effective anti-metastatic, growth-inhibitingchemotherapeutic agent for use in the treatment of cancer.

From the above, it should be clear that the present invention providesan anticancer approach that is reliable for a wide variety of tumortypes, and particularly suitable for invasive tumors. Importantly, thetreatment is effective with minimal host toxicity.

1. An isolated peptide less than 100 amino acids in length, comprisingthe amino acid sequence PHSCN, wherein C is the L-isomer.
 2. An isolatedpeptide less than 100 amino acids in length, comprising the amino acidsequence PHSXN, wherein X is an amino acid selected from the groupconsisting of homo-cysteine, histidine, penicillamine, or the D-isomerof cysteine.
 3. The isolated peptide of claim 1 or 2, wherein the aminoterminus of said peptide is blocked with an acetyl group, and thecarboxy terminus of said peptide is blocked with an amide group.
 4. Apharmaceutical composition comprising a therapeutically effective amountof the peptide of claim 1 or 2, and a physiologically compatibleexcipient.
 5. A method of treating cancer in a subject having cancercomprising administering to the subject a therapeutically effectiveamount of the composition of claim 4, wherein said cancer comprisestumor cells expressing a fibronectin receptor.
 6. The method of claim 5,wherein the cancer is a cancer of the breast or prostate.
 7. The methodof claim 5, wherein the composition is administered intravenously,intrathecally, intraperitoneally, or orally.
 8. The method of claim 5,wherein the subject is a human.
 9. The method of claim 5, furthercomprising administering a cytotoxic agent.